Establishment of cell lines of mammary epithelial origin is critical for studies on the mechanism of action of lactogenic hormones and as a simple, biologically meaningful `in vitro` test system for recombinant DNA constructs destined for expression within the mammary glands of transgenic animals. Milk synthesis occurs within clusters of differentiated mammary epithelial cells and is under the strict control of lactogenic peptides and steroid hormones. Initiation of milk synthesis is thereby coordinated temporally with parturition. The molecular mechanisms by which hormones stimulate and maintain lactation are poorly understood. Post-transcriptional regulation appears to be of primary regulatory significance (Goyette, W. A. et al., Cell (1979) 17:1013; Eisenstein, R. S. and Rosen, J. M., Mol. Cell. Biol.(1988) 8:3183. In mice, prolactin increases the half-life of casein mRNA by some 17 to 25 fold while gene transcription increases by 2 to 4 fold (Goyette, W. A. et al. (1979) idem). Complimentary work with any agricultural dairy species is completely lacking.
Industrial production of proteins using the powerful biosynthetic capacity of the bovine mammary gland is an emerging technology. Mammary epithelial cells within the bovine udder produce over a kilogram of protein daily. This complex protein contains many post-translational modifications. Transgenic animals in which foreign genes are expressed in the mammary gland, offer a method of producing heterologous proteins without the restrictions of prokaryotic fermentation. The major barrier to exploiting this concept are the inadequacies of germ line manipulation in farm animals. Problems include modest expression of transgenes, and poor tissue specific expression. A gene expression screening system which could identify superior gene constructs prior to gene transfer, would improve transgenic animal production.
It would be highly desirable to have established mammary cell lines which demonstrate hormone responsiveness and enhanced secretory capacity. They would offer an unparalleled system to explore novel constructs within the target organ, the mammary gland of livestock species. Large numbers of recombinant DNA constructs can be rapidly assessed at modest cost.
Established cell lines should be clonal and capable of either continuous proliferation or differentiation depending on the hormonal and extracellular matrix signals. A transformed phenotype showing growth in soft agar or tumorigenicity is not desirable. Despite considerable development, no single cell line derived from mammary epithelial presents these attributes. The murine COMMA-D cell line exhibits mammary-specific differentiation when exposed to the appropriate extracellular matrix (ECM) and lactogenic hormones (Eisenstein, R. S. and Rosen, J. M.(1988) idem). However, this cell line is heterogeneous being composed of at least 3 cell types of which only 10-20% are capable of casein synthesis. Subcloning COMMA parental lines leads to a loss of prolactin responsiveness (Medina, D. et al., Exp. Cell. Res. (1987) 172:192) or a loss of ECM requirements and protein secretion (Ball, R. K. et al., Embo. J. (1988) 7:2089).
Spontaneous bovine mammary epithelial cells (BMEC) in long term culture have been reported to differentiate (Schmid, E. et al., J. Cell. Biol. (1983) 96:37). However, lactose and casein secretion is low and not inducible with prolactin (our unpublished data).
A variety of established human breast carcinoma lines exist T97D, MCF-7 (Van Deurs, B. et al., J. Histochem. (1987) 35:461), but these appear to be transformed and show variable expression of differentiate phenotype such polarity, tight functions and secretion of milk specific constituents.
It would be highly desirable to have an immortalized mammary cell line which would have normal physiological responses and hence would not be transformed.
Such a cell line would provide one skilled in the art with an `in vitro` system to study lactation, a gene expression system to screen DNA constructs prior to gene transfer and with a method of indefinitely expressing foreign genes.